In Vivo Tracking And Comparison Of The Therapeutic Effects Of MSCs And HSCs For Liver Injury

Cirrhosis and its related morbidity place a significant burden on health careworldwide. Liver transplantation remains the definitive treatment option for end-stageliver disease. But the mismatch between the number of patients requiring transplantationand the amount of available organs is set to grow, highlighting the need to develop newstrategies to reduce liver scarring and stimulate liver regeneration.More recently, reportsof unexpected plasticity in adult bone marrow have raised hopes that stem cell therapymay offer exciting therapeutic possibilities for patients with chronic liver disease.To date,there are several published human clinical studies investigating the effects of stem celltherapy in patients with liver disease and most of the studies yielded positive results.The cells mostly used to transplant were derived from bone marrow including MSCs,HSCs and unsorted mononuclear cells.Among them, MSCs and HSCs can be obtained in a great quantity. Both stem cells have anti-fibrotic and proregenetative effects in the injuredliver. However, which cell type is more effective in treating the injured liver remains to bedetermined. Moreover, several recent studies have suggested that MSCs and HSCsfunction synergistically for the therapy of diabetes and heart failure and for vascularizingbioengineered tissues. Whether these cells can work synergistically in the injured liver isunclear.In this experiment, the MSCs and HSCs were isolated from the male green fluorescenceprotein transgenic mice. A total of1×106isolated stem cells were resuspended in PBS andslowly infused into the female liver injured mice via the tail vein to evaluate thebiodistribution after the peripheral and the anti-fibrotic activities of these two stem cells.Objectives1. Our aim was to evaluate the biodistribution of the stem cells after the peripheralinfusion of MSCs or HSCs into liver injured mice and compared their homing capacity tothe injured liver.2. To evaluated the anti-inflammatory and anti-fibrotic activities of these two stem cells inthe injured liver and to study whether MSCs and HSCs exhibit synergistic effects intreating liver injury.3. To investigate the underlying molecular mechanisms through which MSCs and HSCsparticipate in injuried liver repair. We hope that these findings contribute to betterunderstanding of the interactions between stem cells and the environment that leads tohoming and integration into livers.Materials and methods1. MSCs were isolated using whole bone marrow culture method and purified usingattachment method. HSCs from bone marrow were sorted by magnetic nanoparticles andflow cytometry using multiple antibody panels. 2. The adhered cells in the first passage and the third passage were observed byfluorescence microscope．Stem cell surface markers were identified by flowcytometer．The cells were also induced to difierentiate into bone and fat cells in vitro.3.After final CCl4injection at3months, the mice except the control group were randomlydivided into different groups to ensure that the relatively level of liver disfunction wasconstant. Mice from the same cohort were randomly allocated to receive different cellstem cells （1x106cells/mice）via injections of the tail vein.4. Distribution of transplanted cells in injuried liver was detected by bio-imaging system,fluorescence assay and Y chromosome sequence.5. Therapeutic potential of transplanted cell for liver cirrhosis was determined by serumassay, survival curve for the liver injured mice and representative photomicrographs ofH&E-stained mouse livers from the different groups.6. Liver fibrosis was quantified with sirius red staining, real-time PCR andimmunofluorescence analysis.7. The characterization of transplanted stem cells in injuried liver was detected byconfocal microscopy assay.8. Hepatocyte regeneration was determined by immunohistochemistry andimmunofluorescence analyses.9. Quantification of the mouse serum levels of growth factors and cytokine weredetermined using enzyme-linked immunosorbent assays kits per the manufacturer’sinstructions.Results1. Isolation and characterization of MSCs and HSCs from GFP transgenic miceThe MSCs were isolated from the bone marrow of GFP-transgenic mice. In the firstpassage, the cells derived from the donors emitted heterogeneous levels of greenfluorescence when observed under the fluorescence microscope and were of various sizes,as observed in bright field. In the third passage, the GFP signal intensity was uniformamong the cells, and the cells exhibited a homogeneous morphology. Flow cytometryanalyses were used to characterize the surface markers of the cultured cells. Most of the cells expressed the standard MSC surface markers, CD90(95.2±6.1%), CD29(85.6±3.5%) and CD105(96.5±4.3%), whereas they were negative for CD45(6.3±2.2%),CD34(7.2±1.8%) and CD80(3.8±1.2%). MSCs could differentiate into osteoblasts andfat cells respectively. HSCs were also successfully isolated by flow cytometry for use inthe following experiments.2. In vivo tracking of MSCs and HSCs.After intravenous infusion, the GFP signals first accumulated in the lung, but by2h,those signals began to decrease, whereas they started to accumulate in the liver and spleenat2h after infusion. During the following hours to days, the GFP signal intensitygradually increased in the liver. From24h to7d, the GFP signal intensity graduallyincreased in the spleen and then decreased. The GFP signal was barely detectable in thekidney. These trends were similar in the MSCs, HSCs and MSCs+HSCs groups. The GFPintensity of the livers in the MSCs group was significantly higher than in the HSCs orMSCs+HSCs group. The number of homing stem cells to the CCl4-induced cirrhotic liverwas significantly higher than that in the normal group. The donor-derived signals werestronger in the livers4w after the cell injection than at2w. These results were confirmedby the percentage of GFP-positive cells detected after nuclear staining with DAPI. From2w on, most of the transplanted cells were located infiltrating the areas around the liver’sportal tracts and interlobular connective tissue. Only a few of the cells migrated toward thecentral region of the hepatic lobes and can be detected in the sinusoids. FACS analyses ofCXCR4expression on MSCs and HSCs shows that CXCR4expressed on MSCs (in thethird passage)(33.2±8.1%) was significantly higher than HSCs(24.5±6.8%, P<0.05).Real-time PCR revealed that CXCR4mRNA expression was higher in MSCs (in the thirdpassage) than in HSCs.3. Comparing the anti-inflammatory and anti-fibrotic activities of transplantatedstem cells in the injured liver.The mice in the normal group survived the observation period. The survival of themice in the three groups that underwent stem cell transplantation was significantly higherthan in the group treated with CCl4. The survival percentage in the MSCs group (68.2%) was significantly higher than in the HSCs (36.4%) or MSCs+HSCs (45.5%) group, whilethe survival percentage in the MSCs+HSCs group was significantly higher than in theHSCs group. The histological sections of the CCl4-injured mice demonstrated that whencompared with normal mice, a large number of inflammatory cells had infiltrated thesinusoids and centrilobular regions, and the coagulation necrosis of hepatocytes wasobserved (H&E staining). In addition, liver fibrosis had increased significantly,characterized by fibrotic septum formation starting in the portal areas (Sirius red staining).After transplantation of MSCs, the injured livers showed maximal restoration with thinnerfibrotic areas and decreased collagen depositions (3.6±0.7%). However, the fibrotic areashad decreased to a lesser extent in the mice transplanted with HSCs (7.6±0.8%) orMSCs+HSCs (6.2±1.7%, P<0.05). These results were confirmed by the expression oftype I collagen (4.0±1.1%,6.3±3.1%,5.7±1.5%, respectively, P<0.05). Moreover, whencompared with the mice in the HSCs group and the MSCs+HSCs group, the micereceiving MSCs showed the best improvement of liver function, however, liver function inMSCs group was still inferior than in the normal cohort, as demonstrated by the ALB,ALT and AST levels of the peripheral blood.4. The mechanism of transplanted MSCs and HSCs promoting liver function andreversing liver fibrosis.The immunofluorescence staining in the HSCs group revealed a higher percentage ofdouble-labeled GFP+/AFP+and GFP+/ALB+cells in the host livers (4.3±0.6%and3.5±0.7%, respectively), compared with the MSCs (1.4±0.5%and2.1±0.3%, respectively)or the MSCs+HSCs (2.8±0.4%and2.4±0.6%, respectively,P<0.05) groups. Theexpression of α-SMA was significantly decreased in the livers of mice transplanted withany cell type. The α-SMA expression in the MSCs group was significantly lower than inthe other two groups. Double-labeled GFP+/α-SMA+cells were not found in any of thegroups. To evaluate whether stem cell transplantation enhances the proliferation ofhepatocytes in cirrhotic livers, the PCNA and Ki-67expression levels were assessed byimmunofluorescence and immunohistochemistry. In the MSCs-transplanted livers, thepercentage of PCNA+(11.5±3.4%) and Ki-67+(8.2±2.7%) cells was increased significantly when compared with those of the HSCs (6.9±1.8%,6.0±1.1%, respectively) andMSCs+HSCs (8.5±3.1%,7.2±1.9%, respectively, P<0.05) groups.Four weeks after cell transplantation, the serum showed a significant increase in NGFin the MSCs (101±12pg/ml) group when compared with the HSCs and MSCs+HSCsgroups (53±5pg/ml and69±7pg/ml, respectively, P<0.05). The levels of HGF and VEGFwere not significantly different among the cell transplantation groups. The expression ofIL-10in the MSCs group (98±22pg/ml) was higher than in the other cell transplantationgroups (HSCs and MSCs+HSCs groups:42±14pg/ml,66±21pg/ml, respectively) or theCCl4group (21±4pg/ml, P<0.05), whereas the concentration of IL-6(45±15pg/ml) andTNF-α (140±75pg/ml) in the MSCs group was lower than that of HSCs (IL-6:77±13pg/ml, TNF-α:227±87pg/ml) and MSCs+HSCs (IL-6:60±9pg/ml, TNF-α:183±15pg/ml,P<0.05) groups.Conclusions1. When compared with HSCs alone and a combination of HSCs and MSCs, MSCs hadthe greatest homing capability to the injured liver.2. MSCs showed the greatest capability to restore the injured liver in vivo and facilitatemice survival when compared with other two groups.3. MSCs exhibited more remarkable paracrine effects and immunomodulatory propertieson hepatic stellate cells and native hepatocytes in the treatment of the liver pathology.4. Synergistic actions of MSCs and HSCs were most likely not observed because the stemcells in liver were detected mostly as single cells, and single MSCs are insufficient toprovide a beneficial niche for HSCs.